In eubacteria, peptide synthesis is initiated at methionine start codons which are read by N-formyl methionine tRNA. Prior to translation initiation the methionyl moiety of the charged tRNA is N-formylated by the action of Met-tRNAi transformylase (E.C.2.1.2.9). The N-formyl group is removed from the native protein by polypeptide deformylase (E.C. 3.5.127), and the initiator methionine can then be cleaved off by methionine aminopeptidase, completing the primer methionine cycle. In contrast, archaea and eukaryotes have a primer methionine cycle devoid of N-formylating and deformylating activities (for review see Mazel, D., Pochet, S. and Marliere, P.: Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13 (1994) 914-923, and Mazel et al., 1996).
Expression of eukaryotic proteins in eubacterial hosts often results in the production of recombinant proteins that retain an N-terminal forrnylrnethionyl residue (examples include bovine somatotropin (Bogosian, G., et al., (1989) Biosynthesis and incorporation into protein of norleucine by Escherichia coli J. Biol. Chem. 264:531-539); eel growth hormone (Sugimoto, S., Yamaguchi, K. and Yokoo, Y.: Isolation and characterization of recombinant eel growth hormone expressed in Escherichia coli. J. Chromatog. 515 (1990) 483-494); human granulocyte colony-stimulating factor (Clogston, C. L., Hsu, Y. R, Boone, T. C. and Lu, H. S.: Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping. Anal. Biochem. 202 (1992) 375-383); bovine fatty acid-binding protein (Specht, B., Oudenampsen-Kruger, E., Ingendoh, A., Hillenkamp, F., Lezius, A. G. and Spener, F.: N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli. J. Biotechnol. 33 (1994) 259-269); bovine cytochrome P450 (Dong et al FASEB J. 9 (1995) A1486); Methanothermus fervidus histone A (Sandman, K., Grayling, R. A., and Reeve, J. N. Improved N-terminal Processing of Recombinant Proteins Synthesized in Escherichia coli. Biotechnology 13 (1995) 504-506); human interleukin-5 (Rose, K., Regarney, P., Anderegg, R, Wells, T., Proudfoot, A., Human interleukin-5 expressed in Escherichia coli has N-terminal modifications. Biochem J. 286 (1992) 825-828); human parathyroid hormone (Rabbani, S. A., Yasuda T., Bennett H. P. J., Sung, W. L. Zahab, D. M., Tam, C. S. Goltman, D., and Hendy, G. N. Recombinant Human Parathyroid Hormone Synthesized in Escherichia coli. Journal of Biological Chemistry 263:3 (1988) 1307-1313; Hogset, A., Blingsmo, O. R., Gaurvk V. T., Saether, O., Jacobsen, P. B., Gordeladzo, J. O., Alestrom, P. and Gautvik, K. M. Expression of Human Parathyroid Hormone In Escherichia coli Biochemical and Biophysical Research Communications 166:1 (1990) 50-60); human gamma-interferon (Honda, S., Asano, T., Kajio, T., and Nishimura, O. Escherichia coli-Derived Human Interferon-.gamma. with Cys-Tyr-Cys at the N-Terminus is Partially Nα-Acylated. Archives of Biochemistry and Biophysics 269 (1989) 612-622)). In addition retention of N-formyl methionine has been found in endogeneous E. coli proteins (Hauschild-Rogat, P. N-formylmethionine as a N-terminal group of E. coli ribosomal protein. Mol. Geri. Genet. 102 (1968) 95-101, Marasco, W. A., Phan, S. H., Kruusch, H., Showell, H. J., Feltner, D. E., Nairn, R, Becker, E. L. and Ward, P. A.: Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotaetic factor produced by Escheriehia coli. J. Biol. Chem. 259 (1984) 5430-5439; Milligan, D. L. and Koshland, Jr., D. E.: The amino terminus of the aspartate chemoreceptor is formylmethionine. J. Biol. Chem. 265 (1990) 4455-4460). Since N-formylated peptides are a major indicator of eubacterial infections for the mammalian immune system and are highly immunogenic, incomplete deformylation precludes; for example, the use of N-formylated preparations for therapeutic purposes. Several approaches to circumvent this problem have been proposed, e.g., expression in the presence of trimethoprim and thymidine (Sandman et al., 1995), overexpression of peptide deformylase in the host (Warren, W. C., Bentle, K. A., Schlittler, M. R, Schwane, A. C., O'Neil, J. P. and Bogosian, G. (1996): increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli. Gene 174, 235-23), expression as a protein fusion either with an N-terminal peptide that can be removed in vitro by a specific protease or with an N-terminal leader peptide which is cleaved during transport of the nascent protein in a non-cytoplasmatic compartment. Finally, the N-formyl group may also be removed by mild acid hydrolysis, or the fraction of the protein retaining N-formyl methionine may be separated from the correctly processed protein by purification procedures.
Each of these approaches has significant disadvantages. Addition of trimethoprim and thymidine is costly, requires manipulation of the culture that will express the recombinant protein, and may slow down growth of the host. Overexpression of peptide deformylase requires a stable plasmid construct in the host that has to be selected for; moreover, deformylation may be less than 100% effective. Expression of fusion proteins requires exact molecular constructions; chemical hydrolysis with acid may cause damage to the rest of the protein. Finally, none of these approaches guarantees a final preparation that is absolutely free of N-formylated peptides derived either from the recombinant protein or from contaminations with endogeneous host peptides.